Amyloid fibrils formation is the well-known hallmark of various neurodegenerative diseases. Thioflavin T (ThT)-based fluorescence assays are widely used to detect and characterize fibrils, however, if performed in bioliquids, the analysis can be biased due to the presence of other, especially abundant, proteins. Particularly, it is known that albumin may bind ThT, although the binding mechanism remains debatable. Here the role of low-order albumin oligomers in ThT binding is investigated using time-resolved fluorometry and size-exclusion chromatography. Under conditions used, the fraction of dimers in human (HSA) and bovine (BSA) serum albumin solutions is as low as ∼7%, however, it is responsible for ∼50% of ThT binding. For both albumins, the binding affinity was estimated to be ∼200 and ∼40μM for monomeric and dimeric species, respectively. Molecular docking suggested that ThT preferentially binds in the hydrophobic pocket of subdomain IB of albumin monomer in a similar position but with a variable torsion angle, resulting in a lower fluorescence enhancement (∼40-fold) compared to amyloid fibrils (∼1000-fold). Dimerization of albumin presumably creates an extra binding site at the subunit interface. These results demonstrate the underestimated role of low-order albumin oligomers that can be highly relevant when analyzing drugs binding using fluorescence spectroscopy.
Keywords: Albumin; Amyloid fibrils; Binding modes; Oligomers; Thioflavin T; Time-resolved fluorometry.
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